The field of this invention is the area of methods for the genetically engineering of non-human animals, in particular those comprising the step of nuclear transfer, and animals produced by such methods.
Techniques for transgenic animal production have been well established for mice (Gordon and Ruddle, 1981) and pigs (Brem et al., 1985; Hammer et al., 1985). However, efficiencies for production of transgenic animals, especially large domestic animals, are generally low. The efficiency of producing a transgenic pig from pronuclear injection can range from 1 to 10% at a cost of approximately $25,000 (Wheeler and White, 1993). The possible use of embryonic stem (ES) cells to produce transgenic animals provides a unique opportunity to increase the efficiency and decrease the cost of transgenic pig production as well as provide more stable gene integration (Wheeler, 1994).
Embryonic stem cells offer an effective tool for producing genetically identical individuals. Embryonic stem cells can be electroporated with deoxyribonucleic acid (DNA) to introduce, replace or inactivate genes of interest. These ES cells can be screened, in vitro, for incorporation of genes in the appropriate location within the genome. Theoretically, screened ES cells could be used as karyoplasts (donor nuclei) for nuclear transfer (NT) to produce an entire individual. This technology has many exciting uses including acceleration of genetic progress, propagation of superior genetic lines and production of transgenic animals. Transgenic animals can be developed for the production of human pharmaceutical proteins, tissues and organs for human transplantation, and animals with improved quantitative traits (i.e., reproduction, growth, carcass or milk composition; Wheeler, 1994). Further, development of NT embryos provides a method for testing the totipotency of ES cell lines.
Nuclear transfer has been reported in amphibians, mice, rats, rabbits, sheep, pigs, cattle (see Prather and First [1990] for review), goats (Yong et al., 1991), domestic cats (Shin et al., 2002), monkeys (Mitalipov et al., 2002), fish (Lee et al., 2002) and mules (Holden, 2003). Numerous studies concerning NT with blastomeres from early embryonic stages have been reported (Prather and First, 1990). However, the number of genetically identical blastomeres is limited with these transfers.
There is a need in the art for efficient means for producing transgenic animals and for reproducing animals, endangered species as well as domesticated livestock.